mmg_233_2014_genetics_genomicsfandomcom-20200215-history
Clostridium difficile
Introduction Clostridium difficile, also known as C. diff, ''is a gram-positive anaerobic bacterium that causes one of the most common nosocomical antibiotic-associated diarrhea in the developed world, which can lead to increased morbidity and mortality. Antibiotic treatment can result in the replacement of normal gut microbiome with ''C. diff, ''which causes colitis or inflammation of the colon. The spore-forming abilities of this bacterium allow it to survive in extreme conditions. When ''C. diff ''releases toxins, bloating, diarrhea, and abdominal pain occur. Due to the increase in severe cases of ''C. diff ''infections (CDI), there has been an increased interest in this bacterium. (1) Pathogenic ''C. diff ''strains produce many toxins. The most widely studied virulence factors are two large clostridial cytotoxins, toxin A (TcdA) and toxin B (TcdB). Both of these toxins are highly potent and cause severe colon inflammation and epithelial tissue damage in the infected hosts, leading to fast fluid loss and diarrhea. The genes that encode for toxin A and B, ''tcdA ''and ''tcdB, are both located in a 19.6 kb region of the PaLoc chromosome, which is known as the pathogenicity locus. (2) Research The importance of toxin A and toxin B individually as virulence factors in the pathogenicity of C. diff has been much debated. A study by Kuehne et al. in 2010 used ClosTron gene knockout system to inactivate genes that produce the two toxins resulting in 2 single mutants (A-B+, A+B-) and double mutant A-B-. This system inactivates genes by insertion of an intron into the protein-encoding region of a gene, which results in a non-functional protein. The genotype of each toxin mutant was determined using polymerase chain reaction (PCR) and DNA sequence analysis in order to confirm where each intron was inserted. Western blot analysis indicated the phenotypes of each strain using toxin-specific antibody probes. TcdA knockouts did not produce toxin A and TcdB knockouts did not produce toxin B. The cells used for i''n vitro'' cell cytotoxicity assays were HT29 (human colon carcinoma) cells and Vero (African green monkey kidney) cells. Both cells respond to toxin A and B with different sensitivities. HT29 cells are more sensitive to toxin A while Vero cells are more sensitive to toxin B. The toxins cause both these cells to die by causing them to lose their morphological shapes or "round." This can be easily seen on light microscopes. To keep the data objective, the researchers measured end-point titre of each series of dilutions. End-point titre is the first of a series of dilutions when the HT29/Vero cell shape appears identical to the morphology of negative controls. After cytotoxicity was measured in vitro, virulence was measured in vivo ''using the hamster model of infection. Each of the 3 mutants including the A+B+ strain were given to eight hamsters. All hamsters were colonized with a ''C. diff 630 strain 1-3 days after inoculation. Symptoms of CDI appeared in those that were inoculated with the A+B+ strain, the A-B+ mutant strain, and A+B- mutant strain with an average death time of 1 day. None of the double negative mutant A-B- hamsters developed any symptoms of CBI in 14 days. The results of the study are that gene A and B knockout showed no virulence while production of toxin A and B by gene A and gene B individually or together both resulted in cytotoxic activity in ''vitro, which led to virulence'' in vivo''. Both toxin A and toxin B play important roles in CBI because each toxin individually causes virulence. This supports previous studies and indicates that earlier studies indicating that toxin A alone is the cytotoxin that causes CBI are wrong. References 1. Wikipedia article: Clostridium difficile colitis. Date accessed: Dec 6, 2014. 2. Carter, GP et al. (Jan 2012). "The role of toxin A and toxin B in the virulence of Clostridium difficile." Trends Microbiology. 20(1): 21-0. 06 Dec. 2014. PMID: 22154163. 3. Kuehne, SA et al. (Oct 2010). "The role of toxin A and toxin B in Clostridium difficile infection." Nature 467 (7316): 711–3. 06 Dec. 2014. doi:10.1038/nature09397. PMID 20844489.